Structural basis for dual roles of Aar2p in U5 snRNP assembly

Abstract

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p-RNase H platform. Known Brr2p interaction sites of the Jab1/ MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phosphomimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly.[Keywords: assembly chaperone; pre-mRNA splicing; regulation by phosphorylation; snRNP biogenesis and recycling; X-ray crystallography] Supplemental material is available for this article. Uridine-rich (U) small nuclear ribonucleoprotein particles (snRNPs) are the main subunits of spliceosomes, the large and dynamic RNP machineries required for the removal of noncoding introns from eukaryotic precursor messenger RNAs (pre-mRNAs) and the ligation of neighboring coding exons. The major spliceosome is built from the U1, U2, U4, U5, and U6 snRNPs, each of which contains an individual small nuclear RNA (snRNA), seven common Sm proteins (or like-Sm [LSm] proteins in the case of U6), and a variable set of particle-specific proteins (for review, see Will and Lü hrmann 2001).A hallmark of the spliceosome is its stepwise assembly from snRNPs and many non-snRNP factors only in the presence of a substrate pre-mRNA (for review, see Wahl et al. 2009). During this process, the spliceosome is repeatedly remodeled with the help of eight highly conserved DEXD/H-box ATPases/RNA helicases (for review, see Staley and Guthrie 1998). Among these enzymes, the U5-specific Brr2 protein is required for the catalytic activation of an initial inactive spliceosomal assembly and again during the ordered disassembly of the postsplicing complex (for review, see Hahn and Beggs 2010). Brr2p is regulated by two other U5 snRNP proteins: Prp8p, considered the master regulator of the spliceosome (for review, see Grainger and Beggs 2005), and Snu114p, a complex G protein that resembles the ribosomal translocase eEF2 (Bartels et al. 2002(Bartels et al. , 2003Brenner and Guthrie 2005;Small et al. 2006).The Sm-type snRNPs themselves are also assemb...

Novel regulatory principles of the spliceosomal Brr2 RNA helicase and links to retinal disease in humans

“…53,54 Structural analyses have shown that Aar2 contains a globular portion and a flexible, C-terminal extension. 41,55,56 The globular part of Aar2 binds the linker, endonuclease, and RH domains of Prp8, while its C-terminal extension spirals along the Prp8 reverse transcriptase domain and across the RH domain, where it connects an extended hairpin of the RH domain with the β-barrel core of the Jab1 domain. 55,56 The Prp8 Jab1 domain is thereby bound to the RH domain in a manner that sterically precludes binding of Brr2.…”

“…41,55,56 The globular part of Aar2 binds the linker, endonuclease, and RH domains of Prp8, while its C-terminal extension spirals along the Prp8 reverse transcriptase domain and across the RH domain, where it connects an extended hairpin of the RH domain with the β-barrel core of the Jab1 domain. 55,56 The Prp8 Jab1 domain is thereby bound to the RH domain in a manner that sterically precludes binding of Brr2. 55,56 Phosphorylation of Aar2 at serine 253, possibly aided by additional phosphorylation at other residues, leads to a conformational change that reduces its affinity for Prp8, and thereby allows Brr2 to bind.…”

Dramatically reduced spliceosome in Cyanidioschyzon merolae

“…Another category of splicing factors that appear to be nearly absent in C. merolae is snRNP biogenesis proteins, the factors involved in initial assembly of snRNPs. The U2 protein Cus2, Aar2 from the cytoplasmic form of U5, Snu40 from the nuclear U5, Sad1 from the tri-snRNP, and the SMN protein required for correct Sm ring assembly in humans were not detected in C. merolae (59)(60)(61). SMN may be rendered unnecessary by the absence of stem-loops 3′ of the Sm binding site in C. merolae snRNAs.…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…However, initial assembly of yeast U5 snRNP in the cytoplasm leads to a pre-U5 particle that contains the assembly factor Aar2 instead of Brr2. [34][35][36][37][38][39] Thus, Brr2 is transported to the nucleus independent of other U5 snRNP components 35 and its helicase activity may have to be shut off during this phase to avoid detrimental off-target effects. Once assembled in the nucleus, mature U5 snRNP joins the U4/U6 di-snRNP to form the U4/U6 U5 trisnRNP, in which Brr2 already encounters its U4/U6 di-snRNA substrate before incorporation into the spliceosome.…”